CN107224578B

CN107224578B - HIV vaccine and preparation method thereof

Info

Publication number: CN107224578B
Application number: CN201710431893.9A
Authority: CN
Inventors: 姜世勃; 陆路; 王茜; 代嫣嫣
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2017-06-09
Filing date: 2017-06-09
Publication date: 2021-01-08
Anticipated expiration: 2037-06-09
Also published as: CN107224578A

Abstract

The invention belongs to the field of vaccines, and relates to an AIDS combined vaccine based on a conformation induction and specific epitope boosting immunization strategy and a preparation method thereof. More particularly, the invention relates to an AIDS combined vaccine, which comprises a DNA vaccine, an envelope protein vaccine and a polypeptide vaccine of a specific site which are used in combination. The combined vaccine can induce and recognize IgG antibody production of natural antigen related to antiviral protection rate to be enhanced, reduce IgA level competing with the IgG, and obviously improve ADCC effect of killing infected target cells, thereby achieving excellent effect of preventing AIDS.

Description

HIV vaccine and preparation method thereof

Technical Field

The invention belongs to the field of vaccines, and particularly relates to a novel AIDS vaccine based on a conformation induction and specific epitope selection boosting strategy and a preparation method thereof.

Background

Acquired Immune Deficiency Syndrome (AIDS) is an acquired immunodeficiency syndrome (AIDS) caused by massive death of Human lymphoid CD4T cells as a result of infection with Human Immunodeficiency Virus (HIV). After the first AIDS case was discovered in 1981, AIDS rapidly spreads worldwide. According to the estimation of the AIDS planning administration of the United nations, about 3670 ten thousand AIDS virus infectors are still existed in the whole world by 2015. In the same year, about 200 million people newly infect the HIV, and 120 million people die from the HIV-related reasons, so that the human health in the world is seriously threatened, the social progress and the economic growth are seriously damaged, and the method is one of the major challenges facing the world. How to effectively prevent HIV infection and reduce morbidity and mortality is an urgent task facing China and even the world.

Vaccines are an effective means of controlling HIV transmission and infection. Unfortunately, no effective vaccine is currently approved for marketing. RV144 is the only vaccine which is proved to have a certain protective effect in clinical tests at present, and the clinical tests show that the vaccine has 31.2 percent of protective effect (3). The reasons for the protective effect of the subsequent immunoassay (4) are mainly: 1) the anti-HIV protection rate is positively correlated with the content level of IgG antibody which recognizes the gp120V1V2 region of HIV envelope protein in a vaccine recipient; 2) the protection against HIV is inversely related to the level of plasma recognition of the HIV envelope protein IgA in vaccine recipients. The IgA in plasma may compete with the IgG to bind to envelope protein, so that the IgG is prevented from playing an antibody-dependent cell-mediated cytotoxicity (ADCC), the killing effect on HIV infected cells is reduced, and the protective effect of the vaccine is weakened. Furthermore, the ADCC effect of IgG directed to the V1V2 region is considered to be closely related to the protective effect of RV144, and may play a key role. Therefore, how to induce higher level of specific IgG for recognizing HIV-1 native conformation V1V2 and ADCC capability mediated by the IgG, and reduce the stimulation of producing IgA for recognizing envelope protein is one of the key technical bottlenecks in HIV vaccine design.

Previous studies have shown that when animals are immunized with pseudoviruses, the immunized antibodies can recognize both the viral envelope protein and a polypeptide containing an epitope sequence. However, antibodies obtained by immunizing animals with a polypeptide having an epitope sequence as an antigen recognize only the polypeptide antigen, and it is extremely difficult to recognize viral envelope proteins having a native conformation (FIG. 1). Therefore, it is very difficult to induce the production of antibodies recognizing envelope proteins of viruses in a natural conformation by simply using polypeptide immunization, which is a common recognition in the current HIV vaccine research. This may be due to the fact that the same sequence has a different conformation on the polypeptide than the native conformation on the virus.

The current design scheme of epitope vaccine using polypeptide as antigen faces several important problems: 1) the epitope polypeptide has relatively small molecular weight and weaker immunogenicity; 2) because polypeptide antigens only contain the amino acid sequence of a key epitope and have no more peripheral auxiliary structures, the polypeptide antigens are difficult to have a natural conformation similar to the epitope on viruses, and therefore, some polypeptide antigens are difficult to induce antibodies capable of recognizing the corresponding epitope in the natural conformation. For example, previous studies have shown that using epitope polypeptides of the MPER region on HIV-1gp 41, antibodies induced to recognize only the epitope polypeptide, but have poor recognition of HIV and no neutralizing activity (1, 2). Therefore, how to use epitope vaccines to induce antibodies that recognize the corresponding epitopes in the virus in their native conformation is a challenge in the design of HIV vaccines.

Disclosure of Invention

Aiming at the two technical difficulties, the invention innovatively provides a method of conformation induction and specific epitope screening to solve the problem of low protection effect of the existing HIV vaccine. The design of conformation induction and specific epitope selection for enhancing immunity provided by the invention can effectively solve the problem that the linear epitope vaccine is difficult to induce and recognize the antibody of the epitope with natural conformation, and enhance the antiviral protection effect of the vaccine.

Thus, in one aspect, the present invention relates to an aids combination vaccine comprising the components separately inoculated in the following order:

(a) a DNA expressing a protein comprising an HIV envelope protein or an amino acid sequence having the same immunogenicity and at least 80% homology to said HIV envelope protein;

(b) a protein comprising an HIV gp120 protein sequence or an amino acid sequence having the same immunogenicity as the HIV gp120 protein and at least 80% homology; and

(c) a polypeptide comprising the HIV gp120V1V2 region or an amino acid sequence that is immunogenic and at least 80% homologous to said HIV gp120V1V2 region.

In a second aspect of the present invention, there is provided a kit for the prevention of aids, comprising:

(1) the invention provides an aids combination vaccine of the first aspect; and

(2) instructions for administering said combined AIDS vaccine.

In a third aspect of the present invention, there is provided the use of the aids combination vaccine of the first aspect of the present invention in the preparation of a medicament for the prevention of aids.

In a fourth aspect of the present invention, there is provided a method of vaccinating a subject for the prevention of aids, comprising the steps of:

i. subjecting said subject to a primary immunization (priming) with an immunologically effective amount of component (a) of the AIDS combination vaccine of the first aspect of the invention and optionally a pharmaceutically acceptable adjuvant;

subjecting said subject to a first booster immunization, preferably two weeks after the initial immunization (priming), with an immunologically effective amount of component (b) of the AIDS combination vaccine of the first aspect of the invention and optionally a pharmaceutically acceptable adjuvant; and is

Subjecting said subject to a second booster immunization with an immunologically effective amount of component (c) of the AIDS combination vaccine of the first aspect of the invention and optionally a pharmaceutically acceptable adjuvant, preferably two weeks after the first booster immunization.

The conformation induction and specific epitope selection immune enhancement strategy of the invention obviously improves the level of specific IgG antibody of HIV-1gp 120V1V2 region in natural conformation, and effectively reduces the generation of IgA for recognizing HIV-1Env in the plasma of immune animals; on the basis, the ADCC effect is remarkably improved (figure 7, figure 8).

Drawings

FIG. 1 is a diagram illustrating the technical difficulties encountered with prior art polypeptide vaccines.

FIG. 2 is a schematic representation of the design principle of the conformation-induced plus specific epitope-selective boosting of the present invention.

FIG. 3 is a bar graph showing recognition of JRFL-V1V2 Ab (purified V1V2 specific antibody after JR-FL pseudovirus immunization) and PEP-V1V2 Ab (purified V1V2 specific antibody after V1V2 polypeptide immunization) with V1V2 polypeptide.

FIG. 4 is a graph showing that JRFL-V1V2 Ab recognizes HIV-1 envelope protein in the native conformation expressed by the cell, while PEP-V1V2 Ab does not recognize HIV-1Env in this native conformation.

FIG. 5 is a bar graph showing that the immune strategy of conformation-induced plus specific epitope-selective boosting increases the level of IgG recognizing the HIV-1V 1V2 region in its native conformation.

FIG. 6 is a bar graph showing that the immune strategy of conformation induction plus specific epitope selection boost reduces the level of IgA that recognizes native HIV-1 Env.

Figure 7 is a bar graph showing that the specific antibodies obtained from the immunization strategy of conformation induction plus specific epitope selection boost have a stronger ADCC effect activation.

Figure 8 is a bar graph showing that specific antibodies obtained from an immunization strategy with conformational induction plus specific epitope selective boosting have the effect of inducing greater ADCC.

Detailed Description

All publications mentioned in this disclosure are herein incorporated by reference in their entirety for the purpose of describing and disclosing the methodologies described in the publications that might be used in connection with the description herein. The publications discussed in the context of the present application are provided solely for their disclosure prior to the filing date of the present application. Moreover, with respect to similar or identical terms in the incorporated references and terms explicitly defined in this disclosure, the term definitions provided in this disclosure shall control in all respects.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Exemplary methods, devices, and materials are described herein, but methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions.

In this context, the term "comprising" is intended to be open-ended, meaning that other components may be included in addition to the components listed after the term, provided that the other components do not interfere or do not significantly interfere with the desired technical effect. Similar expressions may be, for example, "comprise", "consist essentially of … …", "consist essentially of … …". In addition, the term "comprising" may also include only the components listed thereafter, and similar expressions may be, for example, "consisting of … …", "including only".

In this context, the term "polypeptide" is used in its conventional meaning, i.e. as an amino acid sequence. The polypeptide is not limited to a particular length of product. Both peptides and proteins are included within the definition of polypeptide, and the three terms may be used interchangeably herein unless specifically indicated otherwise.

It is known in the art that a wide variety of lymphocyte clones exist in animals or humans. Immunization with different antigens enables the generation of multiple lymphocyte clones secreting antibodies against said different antigens. In particular, the Human Immunodeficiency Virus (HIV) to which the invention relates, (a) when immunised with the V1V2 polypeptide, is capable of inducing a lymphocyte clone which recognises the V1V2 polypeptide, however the majority of the antibodies secreted by this lymphocyte clone recognise the V1V2 polypeptide alone and are unable to recognise the native conformation Env expressed on the cell surface. (B) When DNA expressing HIV Env in a natural conformation is used for immunization, various lymphocyte clones secreting antibodies aiming at different epitopes of Env can be induced, even if the same DNA is further used for enhancing immunization, the induced and secreted antibodies still aim at different epitopes of Env, the proportion of lymphocytes capable of secreting antibodies aiming at a certain effective epitope is still low, and even because the epitope is not a dominant epitope, the proportion of specific antibodies after multiple immunizations is lower. (C) When DNA expressing Env in its native conformation is used for immunization, various lymphocyte clones secreting antibodies against different epitopes of Env can be induced, and further, lymphocyte clones secreting antibodies recognizing gp120 monomers can be induced by immunization twice with gp120 monomers. The epitopes on gp120 are much fewer than Env, and therefore the proportion of lymphocytes secreting effective antibodies can be suitably increased, but still lower (fig. 2 (a) to (C)).

The inventor of the application finds a strategy of reinforcing immunity by using conformation induction and specific epitopes by combining the latest progress in the field of AIDS vaccines and the experience of the existing RV144 vaccine, can effectively solve the problems and promote all protection related factors prompted by the RV144 vaccine.

The strategy proposed by the present inventors for conformational induction plus specific epitope selection boosting is as follows: first a primary immunization with DNA expressing Env in its native conformation, followed by a booster immunization with gp120 monomeric protein, and finally a further selection and boosting with V1V2 polypeptide (fig. 2 (D)).

Specifically, in the present invention, the inventors can induce the production of antibodies recognizing Env in the native conformation by primary immunization using DNA, such as a plasmid, capable of expressing Env in the native conformation. Subsequently, the gp120 protein is used for boosting, thereby stimulating the production of more gp 120-specific antibodies. Finally, the polypeptide containing the V1V2 region is utilized to strengthen and induce the immune response generated by the antibody aiming at the V1V2 region, so that the specific antibody for recognizing the natural conformation V1V2 can be generated, the quantity and the proportion are selectively improved, and the aim of stimulating and enhancing the generation of the specific effective antibody is fulfilled.

The experimental results in the present invention show that the production of specific IgG antibodies recognizing the V1V2 region in the native conformation is significantly increased by the novel strategy in the present invention, effectively reducing the level of IgA recognizing Env in the plasma of immunized animals (fig. 5-6). On the basis, the ADCC effect can be obviously improved (figures 7-8). ADCC refers to antibody-dependent cell-mediated cytotoxicity, which is considered to be a key factor in the protective effect of RV144 vaccines (5).

Accordingly, in one aspect, the present invention relates to an aids combination vaccine comprising components which are separately inoculated in the following order:

(a) a DNA expressing a protein comprising an HIV-1 envelope protein or an amino acid sequence having the same immunogenicity and at least 80% homology to said HIV envelope protein;

(b) a protein comprising an HIV-1gp120 protein sequence or an amino acid sequence that is immunogenic and at least 80% homologous to said HIV gp120 protein sequence; and

(c) a polypeptide comprising the HIV-1gp 120V1V2 region or an amino acid sequence having the same immunogenicity and at least 80% homology to said HIV gp120V1V2 region.

In one embodiment, the HIV virus may be HIV-1 or HIV-2, preferably HIV-1.

Herein, the term "protein comprising an HIV envelope protein or an amino acid sequence having the same immunogenicity and at least 80% homology to said HIV envelope protein" refers to an amino acid sequence which, in addition to the HIV envelope protein or the amino acid sequence itself having the same immunogenicity and at least 80% homology to said HIV envelope protein, optionally further comprises other amino acid sequences which do not affect the expression and immunogenicity of said HIV envelope protein or the amino acid sequence having the same immunogenicity and at least 80% homology to said HIV envelope protein. The amino acid sequences that can be included can be readily determined by one of ordinary skill in the art based on knowledge of that person.

Herein, the term "an amino acid sequence having the same immunogenicity and at least 80% homology with the HIV envelope protein" refers to an amino acid sequence having the same immunogenicity and at least 80% homology, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% homology with the HIV envelope protein.

In one embodiment, the sequence of the HIV envelope protein is SEQ ID NO 1, as shown below:

MRVKGIRKSYQYLWKGGTLLLGILMICSAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRLISCDTSVITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAIEAQQRMLQLTVWGIKQLQARVLAVERYLGDQQLLGIWGCSGKLICTTAVPWNASWSNKSLDRIWNNMTWMEWEREIDNYTSEIYTLIEESQNQQEKNEQELLELDKWASLWNWFDITKWLWYIKIFIMIVGGLVGLRLVFTVLSIVNRVRQGYSPLSFQTLLPAPRGPDRPEGIEEEGGER。

herein, the term "protein comprising an HIV gp120 protein or an amino acid sequence having the same immunogenicity and at least 80% homology to said HIV gp120 protein" refers to an amino acid sequence which, in addition to the HIV gp120 protein or the amino acid sequence itself having the same immunogenicity and at least 80% homology to said HIV gp120 protein, optionally further comprises other immunogenicity which does not affect the HIV gp120 protein or the amino acid sequence having the same immunogenicity and at least 80% homology to said HIV gp120 protein. The amino acid sequences that can be included can be readily determined by one of ordinary skill in the art based on knowledge of that person.

Herein, the term "an amino acid sequence having the same immunogenicity and at least 80% homology to the HIV gp120 protein" refers to an amino acid sequence having the same immunogenicity and at least 80% homology, e.g. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% homology to the HIV gp120 protein.

In one embodiment, the HIV gp120 has the sequence of SEQ ID NO 2, as shown below:

LWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRLISCDTSVITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKR。

herein, the term "polypeptide comprising the HIV gp120V1V2 region or an amino acid sequence having the same immunogenicity and at least 80% homology to said HIV gp120V1V2 region" refers to an amino acid sequence optionally further comprising, in addition to the HIV gp120V1V2 region or the amino acid sequence itself having the same immunogenicity and at least 80% homology thereto, an additional immunogenicity that does not affect the HIV gp120V1V2 region or an amino acid sequence having the same immunogenicity and at least 80% homology to said HIV gp120V1V2 region. The amino acid sequences that can be included can be readily determined by one of ordinary skill in the art based on knowledge of that person.

Herein, the term "an amino acid sequence having the same immunogenicity and at least 80% homology to said HIV gp120V1V2 region" refers to an amino acid sequence that is at least 80% homologous, e.g. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% homologous to said HIV gp120V1V2 region.

In one embodiment, the sequence of the HIV gp120V1V2 region is SEQ ID NO 3, as shown below:

GTVKLTPLCVTLNCKDVNATNTTNDSEGTMERGEIKNCSFNITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRLISCDTSVITQA。

in a specific embodiment, the sequence of the HIV envelope protein is SEQ ID NO 1, the sequence of the HIV gp120 is SEQ ID NO 2, and the sequence of the HIV gp120V1V2 region is SEQ ID NO 3.

In one embodiment, the DNA expressing a protein comprising an HIV envelope protein or an amino acid sequence that is identical in immunogenicity and at least 80% homologous to the HIV envelope protein is a plasmid expressing a protein comprising an HIV envelope protein or an amino acid sequence that is identical in immunogenicity and at least 80% homologous to the HIV envelope protein, or a virosome comprising an HIV envelope protein or an amino acid sequence that is identical in immunogenicity and at least 80% homologous to the HIV envelope protein. Preferably, the plasmid may be pCDNA-JR-FL-Env, hereafter pEnv, from NIH AIDS Research and Reference Reagent Program, see Wang et al (2010) (8).

In one embodiment, the protein comprising an HIV gp120 protein sequence or an amino acid sequence with the same immunogenicity and at least 80% homology to the HIV gp120 protein comprises only an HIV gp120 protein or an amino acid sequence with the same immunogenicity and at least 80% homology to the HIV gp120 protein. The protein comprising the HIV gp120 protein or an amino acid sequence having the same immunogenicity as the HIV envelope protein and at least 80% homology may be produced by viral isolation, recombinant expression, or synthesis, or may be obtained commercially.

In one embodiment, the polypeptide comprising the HIV gp120V1V2 region or an amino acid sequence that is immunogenic and at least 80% homologous to the HIV gp120V1V2 region may be produced by expression or synthesis in eukaryotic cells, or obtained commercially. For example, the polypeptide comprising the HIV gp120V1V2 region or an amino acid sequence having the same immunogenicity and at least 80% homology as the HIV gp120V1V2 region is obtained by in vitro expression using a vector into which a DNA encoding the polypeptide comprising the HIV gp120V1V2 region or an amino acid sequence having the same immunogenicity and at least 80% homology as the HIV gp120V1V2 region is cloned. The vector may be, for example, pcDNA 3.

In one embodiment, the polypeptide comprising the HIV gp120V1V2 region or an amino acid sequence having the same immunogenicity and at least 80% homology as the HIV gp120V1V2 region may comprise only the HIV gp120V1V2 region or an amino acid sequence having the same immunogenicity and at least 80% homology as the HIV gp120V1V2 region as a direct immunogen or as part of a combined immunogen.

In one embodiment, the aids combination vaccine may further comprise a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvants include, but are not limited to Freund's adjuvant, aluminum hydroxide adjuvant, MF59, AS02A, QA-21, Corynebacterium parvum, lipopolysaccharide, cytokine, alum, and oil adjuvant or water adjuvant. Furthermore, the pharmaceutically acceptable adjuvants used in combination with components (a), (b) and (c) may be the same or different.

The AIDS combined vaccine of the invention can be inoculated by means of injection, such as intramuscular injection, subcutaneous injection, intradermal injection and the like. Those skilled in the art can formulate the antibody to suit the target for specific antibody induction and the corresponding mode of administration.

In a second aspect, the present invention provides a kit for preventing aids, comprising: 1) the combined AIDS vaccine of the first aspect of the invention; and 2) instructions for administering the AIDS combination vaccine. The information of the immunization sequence, the immunization time interval, the immunization effective dose and the like of each component of the AIDS combined vaccine can be noted on the instruction.

In a third aspect, the present invention provides the use of the AIDS combination vaccine according to the first aspect of the invention in the preparation of a medicament for the prevention of AIDS.

In a fourth aspect, the present invention provides a method of immunizing a subject for the prevention of aids, comprising the steps of:

subjecting said subject to a first booster immunization with an immunologically effective amount of component (b) of the AIDS combination vaccine of the first aspect of the invention and optionally a pharmaceutically acceptable adjuvant; and is

Subjecting said subject to a second booster immunization with an immunologically effective amount of component (c) of the AIDS combination vaccine of the first aspect of the invention and optionally a pharmaceutically acceptable adjuvant.

As used herein, an "immunologically effective amount" refers to an amount of vaccine that, upon administration to a subject, is capable of producing neutralizing antibodies in the subject sufficient to prevent a disease, such as AIDS for the present invention.

In one embodiment, the subject may be, for example, a human.

For the above-mentioned primary immunization, primary booster immunization and secondary booster immunization, it may be performed one or more times without specific number of times as long as the desired technical effect is achieved or realized. Preferably, the primary immunization may be performed once, twice or three times, preferably three times. Preferably, the first booster immunization may be performed once or twice, preferably twice. Preferably, the second booster immunization may be performed once or twice, preferably twice.

In a preferred embodiment, the first boost is performed two weeks after the primary immunization.

In another preferred embodiment, a second boost is performed two weeks after the first boost.

As described above, the AIDS combined vaccine of the invention comprises three components which are used in combination, the three components are used for orderly immunizing organisms, the generation of IgG antibody which recognizes the natural conformation V1V2 is enhanced, the HIV-1Env specific IgA level which competes with the IgG is reduced, and the ADCC effect of killing infected target cells is obviously improved, thereby obtaining the more excellent AIDS vaccine effect compared with the prior RV144 vaccine. The method and strategy of the invention can be widely applied to the development of AIDS vaccine and other infectious diseases.

Examples

The invention is further illustrated by the following examples, but any examples or combination thereof should not be construed as limiting the scope or implementation of the invention. The scope of the invention is defined by the appended claims, and the scope defined by the claims will be clearly understood by those skilled in the art from this description and the common general knowledge in the art. Those skilled in the art can make any modification or change to the technical solution of the present invention without departing from the spirit and scope of the present invention, and such modifications and changes are also included in the scope of the present invention.

Example 1 recognition of V1V2 Polypeptides by V1V 2-specific antibodies of different vaccines

Pseudovirus packaging: VigoFect (VigoFect Inc. Bei lacing, China) transfection reagent was used, as indicated, every 9.6cm²The dishes of (5) were transfected with 5. mu.g of JR-FL-Env plasmid (pEnv) and 5. mu.g of pNL4-3 plasmid, respectively. The medium was replaced with fresh medium 8h after transfection. After 3 days, the supernatant was collected, centrifuged at 3000rpm × 10min to remove cell debris, and the supernatant was stored at-80 ℃ for pseudovirus packaging. For a specific preparation method, Sun et al (7) can be referred to.

HIV-1JR-FL pseudovirus is purified by a kit, dialyzed into PBS, and detected and quantified by using a p24 detection kit, wherein the pseudovirus used in each immunization is 16 mu g/mouse, and an equal volume of ODN CpG adjuvant is added. V1V2 polypeptide according to the conventional immunization method, each immunization uses 50. mu.g of polypeptide, and the polypeptide solution is prepared first and mixed and emulsified with equal volume of Freund's adjuvant. JR-FL pseudovirus (prepared as described above) and HIV-1V 1V2 polypeptide (purchased from Jiangsu Haiyuan protein Biotech Co., Ltd., SEQ ID NO:3,the product number is pJW4303/gp70V1V2-JRFL) to immunize New Zealand white rabbits, and the boosting immunization is carried out every two weeks, and blood is taken before each immunization. After 5 immunizations, rabbit sera were collected and antibody titers were determined by ELISA. Total IgG in serum was first extracted using Protein G column (GE Healthcare, Sweden), and V1V 2-specific antibody was then extracted using V1V2 polypeptide-specific conjugate column (GE Healthcare, Sweden) as described. The binding of the purified specific antibody to the V1V2 polypeptide was determined using enzyme-linked immunosorbent assay (ELISA). The specific operation of ELISA was as follows: using coating solution (0.05M NaHCO)₃pH 9.6) the V1V2 polypeptide was diluted to 5. mu.g/ml and coated on 96-well ELISA plates at 4 ℃ overnight at 50. mu.l/well. The coated plate was washed twice with wash solution (0.05% tween in PBS) and then blocked with 2% skim milk solution 200 μ l/well and incubated at 37 ℃ for 2 h. The purified V1V 2-specific IgG antibody was diluted in PBS in a gradient of 50. mu.l system, and then the ELISA plate was added, incubated at 37 ℃ for 1 hour and washed 3 times with a washing solution, and horseradish peroxidase (HRP) -labeled anti-rabbit IgG antibody (Dako, Glostrup, Denmark) (1:2500) was added, incubated at 37 ℃ for 1 hour and washed 5 times with a washing solution, and then developed with a developing solution prepared with Tetramethylbenzidine (TMB).

As shown in FIG. 3, OD of V1V2 polypeptide bound to specific antibody (JRFL-V1V2 Ab) purified after immunization with JR-FL pseudovirus at a concentration of 5. mu.g/ml for V1V 2-specific IgG antibody₄₅₀The value was 1.2, whereas OD of a specific antibody (PEP-V1V2 Ab) purified after immunization with the V1V2 polypeptide bound to the V1V2 polypeptide₄₅₀The value was 1.28. As can be seen, both JRFL-V1V2 Ab and PEP-V1V2 Ab antibodies can well bind to the V1V2 polypeptide.

Example 2 recognition of HIV-1Env in its native conformation by different specific antibodies

5ml of HIV-1JR-FL live virus stock was added to 4X 10⁶CEM. NKR. CCR5 cells, gently flicked about 60 times, and then the cell and virus mixture was CO-washed at 37 deg.C₂Incubate in the incubator for 2h with gently shaking the resuspended cells every 30 min. Then the cell and virus mixture is added to the appropriate volume of the 1640 medium containing 10% FBS, transferred to a culture flask and placed at 37 ℃ in CO₂Culturing in an incubator. In order to ensure the cell metabolism needs and timely fluid infusion,cells at day four post-infection were the target cells (hereinafter referred to as target cells) of cem. nkr. ccr5 expressing HIV-1JR-FL Env (see Bruel et al (6)).

Mu.l each of 10. mu.g/ml JRFL-V1V2 Ab and PEP-V1V2 Ab (containing 1% BSA) was mixed with 10. mu.l⁶Target cells of CEM. NKR. CCR5 were mixed well and incubated at room temperature for 1 h. The cells were then harvested by centrifugation at 1000rpm × 3min, resuspended in 200 μ l PBS, centrifuged to discard the supernatant, and repeated three times. Then 100. mu.l of 546 red fluorescently labeled anti-rabbit IgG antibody (Life Technologies, America) (1:500) was added for resuspension and incubated at room temperature for half an hour. After that, the cells were collected by centrifugation at 1000rpm X3 min, resuspended in 200. mu.l of PBS, centrifuged to discard the supernatant, and repeated three times. The number of cells fluorescently labeled was detected using Accuri C6 (BD).

As shown in fig. 4, JRFL-V1V2 Ab strongly bound to cem. nkr. ccr5 target cells (14.9%), whereas PEP-V1V2 Ab did not substantially bind to cem. nkr. ccr5 target cells (0.6%).

Example 3 comparison of the content of V1V 2-specific IgG recognizing the HIV-1Env in the native conformation in different antibodies of the immune group

New Zealand white rabbits were divided into six groups of three rabbits each, and treated as follows: the first group was immunized five times with PBS, every two weeks, 1 ml/mouse; the second group uses HIV-1gp120 monomer to immunize five times, prepares high-concentration protein solution, uses PBS to dilute the protein solution, then mixes and emulsifies the diluted protein solution with Freund's adjuvant with the same volume, immunizes once every two weeks, immunizes 50 mug/piece each time; the third group is immunized five times by using HIV-1V 1V2 polypeptide (purchased from Jiangsu Haiyuan protein biotechnology limited, the sequence of which is SEQ ID NO:3, the product number of which is pJW4303/gp70V1V2-JRFL), the polypeptide solution is diluted by PBS, and then the diluted polypeptide solution is mixed and emulsified with Freund's adjuvant with the same volume, and the immunization is carried out once every two weeks, wherein 50 mu g/mouse is immunized each time; the fourth group was immunized three times with a plasmid expressing JR-FL Env (pEnv), diluted with PBS to a plasmid solution, then emulsified in an equal volume of Freund's adjuvant mixed, once every three weeks, with 50. mu.g/mouse of immunization, and then boosted four times with HIV-1gp120 monomeric protein, with 50. mu.g/mouse of immunization, once every two weeks; the fifth group was immunized three times with a plasmid expressing JR-FL Env (pEnv), followed by four booster and selection immunizations with the HIV-1V 1V2 polypeptide, as above; in the sixth group, the JR-FL Env expression plasmid (pEnv) is used for immunization for three times, then HIV-1gp120 monomer protein is used for boosting immunization for two times, and finally HIV-1V 1V2 polypeptide is used for boosting and selective immunization for two times, wherein the immunization method is the same as the above.

After immunization was complete, sera were collected and IgG purified, and V1V2 specific IgG antibodies were extracted using V1V2 polypeptide specific conjugate column as described. The antibodies obtained from the aforementioned first to sixth groups were labeled as a PBS group (specific antibody purified after completion of PBS immunization), a gp120 group (specific antibody purified after completion of gp120 monomeric protein immunization), a V1V2 group (specific antibody purified after V1V2 polypeptide immunization), a pEnv + gp120 group (specific antibody purified after immunization with a plasmid expressing JR-FL Env and then enhanced with gp120 monomeric protein), a pEnv + V1V2 group (specific antibody purified after selection immunization and then enhanced with V1V2 polypeptide) and a pEnv + gp120+ V1V2 group (specific antibody purified after selection immunization and then enhanced with JR-FL Env, gp120 monomeric protein and finally V1V2 polypeptide), respectively, in groups.

Mu.l of each of the six antibodies (diluted with 1% BSA) at 10. mu.g/ml were mixed with 10. mu.l of each of the six antibodies⁶Target cells of cem. nkr. ccrr 5 expressing JR-FL Env were mixed and incubated at room temperature for 1 hour. The cells were then harvested by centrifugation at 1000rpm × 3min, resuspended in 200 μ l PBS, centrifuged to discard the supernatant, and repeated three times. Add 100. mu.l 546 red fluorescently labeled anti-rabbit IgG antibody (1:500) for resuspension and incubate at room temperature for half an hour. The cells were then harvested by centrifugation at 1000rpm × 3min, resuspended in 200 μ l PBS, centrifuged to discard the supernatant, and repeated three times. The number of cells fluorescently labeled was detected using Accuri C6 (BD).

As a result, as shown in fig. 5, the antibodies of both gp120 and V1V2 were less likely to bind to JR-FL Env expressed on the surface of cem. nkr. ccrr 5 target cells, which were 21.2% and 10.3%, respectively. The antibody generated after conformation induction and gp120 monomer protein reinforcement by using Env-expressing plasmid primary immunization, namely pEnv + gp120 group antibody, is obviously reinforced by combining with JR-FL Env expressed on the surface of CEM. NKR. CCR5 target cells, and is 46.1%. In addition, the effect of binding JR-FL Env expressed on the surface of CEM.NKR.CCR5 target cells by using an antibody produced by using an Env-expressing plasmid for conformational induction and then directly using a V1V2 epitope polypeptide for selection and reinforcement, namely a pEnv + V1V2 group antibody, is improved compared with the gp120 group and the V1V2 group, but the effect is not obvious and is 32.6%. This is probably because, after conformational induction, although lymphocyte clones secreting antibodies recognizing different epitopes of Env were produced, the number of these clones was so low that induction directly with the specific epitope V1V2 polypeptide did not produce a significant effect. However, after conformational induction by using plasmid expressing Env, the antibody generated after using gp120 monomer protein to strengthen and select lymphocyte clones capable of secreting antibodies recognizing different epitopes of gp120 and then using specific antibody epitope V1V2 polypeptide to further select cells capable of secreting antibodies recognizing epitopes of V1V2 region on gp120, namely the antibody of pEnv + gp120+ V1V2 group, obviously improves recognition of JR-FL natural conformation Env expressed on the surface of CEM.NKR.CCR5 target cells to 65.3%.

Example 4 comparison of IgA content recognizing HIV-1Env in native conformation in different antibodies of the immune group

After completion of the immunization of the above New Zealand white rabbits (example 3), the antibody titer was measured and the serum was collected. IgG and IgA in serum were purified using Protein L/Agarose (Invivo Gen, America) column according to the instructions, and IgG was removed using Protein G column (GE Healthcare, Sweden) to obtain IgA antibody in serum. PBS group (IgA antibody purified after PBS immunization is finished), gp120 group (IgA antibody purified after gp120 monomeric protein immunization is finished), V1V2 group (IgA antibody purified after V1V2 polypeptide immunization), pEnv + gp120 group (IgA antibody purified after immunization is primed with a plasmid expressing JR-FL Env and then boosted with gp120 monomeric protein), pEnv + V1V2 group (IgA antibody purified after selection immunization is primed with a plasmid expressing JR-FL Env and then boosted with V1V2 polypeptide), pEnv + gp120+ V1V2 group (IgA antibody purified after selection immunization is primed with a plasmid expressing JR-FL Env, then boosted with gp120 monomeric protein and finally boosted with V1V2 polypeptide).

The present inventors examined the content of IgA recognizing JR-FL Env in these sera. The method comprises the following specific steps: mixing the above six IgA antibodies 10 μ g/mlMu.l of the mixture (containing 1% BSA) were mixed with 10⁶Target cells of cem. nkr. ccrr 5 were mixed and incubated at room temperature for 1 hour. The cells were then harvested by centrifugation at 1000rpm × 3min, resuspended in 200 μ l PBS, centrifuged to discard the supernatant, and repeated three times. Then 100. mu.l of Fluorescein Isothiocyanate (FITC) -labeled anti-rabbit IgA secondary antibody (Abcam, England) (1:500 dilution) was added to resuspend the cells and incubated at room temperature for half an hour. The cells were harvested by centrifugation at 1000rpm × 3min, resuspended in 200 μ l PBS, centrifuged to discard the supernatant, and repeated three times. The number of fluorescently labeled target cells was detected using Accuri C6 (BD).

As a result, as shown in fig. 6, immunization directly using gp120 monomeric protein (gp120 group) induced high level of IgA (19%) recognizing JR-FL Env expressed on the surface of cem. nkr. ccrr 5 target cells. While the plasmid expressing Env was used for conformational induction and then enhanced by gp120 monomeric protein, IgA bound to target cells of cem. nkr. ccr5 was present in the IgA antibody of the pEnv + gp120 group, but the immunity was reduced to 10.3% compared to the immunization with gp120 monomeric protein alone, which is consistent with clinical data (3). Further, after conformational induction using Env-expressing plasmid priming, selection and boosting using V1V2 epitope polypeptide directly, IgA bound to JR-FL Env expressed on the surface of cem. nkr. ccrr 5 target cells was low, 2%, in the group of pEnv + V1V2 IgA antibodies. This is probably because, after conformational induction, although lymphocyte clones secreting antibodies recognizing different epitopes of Env were produced, these clones were low in number, and in addition the epitope of the V1V2 polypeptide was single, and subsequent immune induction with a specific epitope may result in a lower amount of IgA recognizing Env. However, after conformational induction using Env-expressing plasmid priming, lymphocyte clones secreting antibodies recognizing different epitopes of gp120 were selected by gp120 monomer protein boosting, and then further selected by specific antibody epitope V1V2 polypeptide boosting, the content of IgA recognizing Env expressed on the surface of cem. nkr. ccrr 5 target cells in IgA antibodies of the pEnv + gp120+ V1V2 group was significantly reduced to 3.5%. This is probably due to the low number of lymphocyte clones that, after conformational induction, although capable of producing antibodies that recognize different epitopes of Env; in addition, although immune responses to gp120 could be recognized by gp120 monomeric protein selection, continuing with V1V2 polypeptide selection and boosting, immune responses recognizing other Env and gp120 epitopes could not persist, while immune responses recognizing V1V2 epitopes were enhanced, thus potentially leading to lower amounts of IgA recognizing Env.

Example 5 detection of activation of ADCC Effect by antibodies of different immune groups

After the new zealand white rabbits were immunized (example 3), the antibody titers were measured and sera were collected. IgG was first purified from serum as described in example 1, and then specific antibodies in serum were purified using V1V2 polypeptide-specific conjugate columns as described: the specific antibody (PBS group) obtained after PBS immunization, the specific antibody (pEnv + gp120 group) obtained after the primary immunization by a plasmid (pEnv) expressing the JR-FL Env and the enhancement immunization by the gp120 monomeric protein, and the specific antibody (pEnv + gp120+ V1V2 group) obtained after the primary immunization by the plasmid (pEnv) expressing the JR-FL Env and the enhancement immunization by the gp120 monomeric protein and the enhancement selection immunization by the V1V2 polypeptide. The ability of these specific antibodies to activate the ADCC effect was tested. ADCC is considered to be a key factor in the protective effect of RV144 vaccines, so we chose this index to evaluate the efficacy of the new vaccines designed.

In the invention, an ADCC Reporter Bioassay deployment Model (Promega, Madison, Wis.) of Promega is selected, and the Model can better detect the activation of the ADCC effect by the antibody. The ADCC effect of antibodies was also studied using this model in articles such as the Nature journal (6, 7). The activation ability of the antibody to the ADCC effect can be indirectly detected by detecting the fluorescence value, and the stronger the activation ability to the ADCC effect, the stronger the ADCC effect that is activated by the antibody, and the higher the fluorescence value corresponding thereto.

The specific scheme for detecting the ADCC effect activation capability of the antibody is as follows: after purification of IgG in the serum, V1V 2-specific antibodies were purified using NHS column coupled to V1V2 polypeptide, and filter sterilized for testing for activation of ADCC effect. Monocyte (Jurkat cells) cells were plated in 96-well plates (500,000 cells/well). The target cell is a CEM.NKR.CCR5 target cell expressing HIV-1Env after being infected by HIV-1JR-FL live virus. Target cells (10,000 cells/well) were incubated with varying concentrations of antibody (5. mu.g/ml, 15. mu.g/ml, 30. mu.g/ml) at room temperatureEffector cells were added after 30 min. The cell plate was centrifuged at 300 g.times.5 min and then placed at 37 ℃ in 5% CO₂Incubate for 24 h. The cell supernatant (100. mu.l) was transferred to a blank plate, and an ADCCReporter Bioassay amplification Model (Promega) developing reagent was added at 100. mu.l/well. The fluorescence value of the Infinite M200 Pro microplate reader is detected.

As shown in fig. 7, the induced fluorescence value was higher as the antibody concentration was increased. After direct immunization with PBS, the generated PBS group-specific IgG antibody (with the concentration of 30 mu g/ml) can induce a certain fluorescence value (RLU: 9365), and has extremely weak activation effect on ADCC effect; after conformation induction and gp120 monomeric protein reinforcement, the fluorescence value induced by the generated pEnv + gp120 group specific IgG antibody (the concentration is 30 mu g/ml) is obviously enhanced (RLU: 520131), and the activation effect on ADCC effect is improved; after conformation induction and gp120 monomeric protein reinforcement, and further selection and reinforcement by using V1V2 polypeptide, the fluorescence value induced by the generated pEnv + gp120+ V1V2 group specific IgG antibody (the concentration is 30 mu g/ml) is further improved (RLU: 754498), which shows that the activation effect of the antibody on ADCC effect is obviously enhanced.

Example 6 detection of ADCC Effect on antibodies of different immune groups

After the new zealand white rabbits were immunized (example 3), the antibody titers were measured and sera were collected. IgG was first purified from serum as described in example 1, and then specific antibodies in serum were purified using V1V2 polypeptide-specific conjugate columns as described: the specific antibody (PBS group) obtained after PBS immunization, the specific antibody (pEnv + gp120 group) obtained after the primary immunization by a plasmid (pEnv) expressing the JR-FL Env and the enhancement immunization by the gp120 monomeric protein, and the specific antibody (pEnv + gp120+ V1V2 group) obtained after the primary immunization by the plasmid (pEnv) expressing the JR-FL Env and the enhancement immunization by the gp120 monomeric protein and the enhancement selection immunization by the V1V2 polypeptide. The ADCC effect mediated by these specific antibodies was examined. ADCC is considered to be a key factor in the protective effect of RV144 vaccines, so we chose this index to evaluate the efficacy of the new vaccines designed.

The specific scheme for detecting the ADCC effect of the antibody is as follows: purification of IgG in serum followed by conjugation with V1V2Peptide NHS column V1V 2-specific antibodies were purified, filter sterilized for ADCC assay. Peripheral blood mononuclear cells (PBMC, whole cells) were plated in 96-well plates (500,000 cells/well), incubated at 37 ℃ for 3h to allow cells to adhere, and then washed twice with incubated PBS to remove nonadherent cells, with the adherent cells as effector cells. The target cell is a CEM.NKR.CCR5 target cell expressing HIV-1Env after being infected by HIV-1JR-FL live virus. Cem. nkr. ccr5 target cells (10,000 cells/well) and different concentrations of antibody (1.67 μ g/ml, 5 μ g/ml, 15 μ g/ml) were incubated at room temperature for 30min before effector cells were added. The cell plate was centrifuged at 300 g.times.5 min and then placed at 37 ℃ in 5% CO₂Incubate for 12 h. Cell supernatants (100. mu.l) were transferred to blank plates and CytoTox-ONE was added^TMChromogen Membrane integration Assay (Promega) 100. mu.l/well. The lactate dehydrogenase produced by the cleaved cellular Lactate Dehydrogenase (LDH) is converted to a cytochrome oxidase substrate to produce fluorescence (Resazurin), which is detected by a fluorometer (Ex 560nm/Em 590 nm). Calculating the proportion of specific killing: (experimental-blank)/(positive-blank). times.100%. Target cells were treated with 2. mu.l of lysis buffer (Cytotox-ONE Homogeneous Membrane integration Assay Kit) alone as a positive control, and target cells and effector cells were treated without antibody as a negative control. As shown in fig. 8, the induced fluorescence value was higher as the antibody concentration was increased. After direct immunization with PBS, the proportion of target cells killed by the generated PBS group-specific IgG antibody (concentration 15 μ g/ml) was 0.6654%, mediating a very weak ADCC effect; after conformation induction and gp120 monomeric protein reinforcement, the ratio of the generated pEnv + gp120 group specific IgG antibody (the concentration is 15 mu g/ml) to kill target cells is 14.03 percent, which indicates that the ADCC effect induced by the antibody is improved; after conformation induction and gp120 monomeric protein reinforcement, the V1V2 polypeptide is used for further selection and reinforcement, the ratio of the generated antibody pEnv + gp120+ V1V2 group specific IgG antibody (the concentration is 15 mu g/ml) to kill target cells is 19.73 percent, which indicates that the ADCC effect induced by the antibody is obviously enhanced.

The detection result of ADCC effect is synthesized, which is probably because the content of V1V2 specific IgG for recognizing HIV-1Env in a natural conformation is increased after conformation induction of pEnv immunity and gp120 monomer strengthening immunity (figure 5), so that the ADCC effect activating capability and mediated ADCC effect are improved; after further selection and boosting by using the V1V2 polypeptide, the IgG content of the pEnv + gp120+ V1V2 group-specific IgG antibody for recognizing the native conformation is remarkably increased, and the ADCC effect activating capability and mediated ADCC effect are also remarkably enhanced. The index shows that the vaccine designed by the inventor can induce antibodies with stronger ADCC effect in vivo.

It will be obvious to those skilled in the art that the invention is not limited to the details of the foregoing examples, and that variations of the embodiments described herein are possible without departing from the spirit or scope of the invention as defined by the appended claims. The scope of protection of the invention is defined by the appended claims.

In addition, it should be understood that although the present invention has been described in terms of embodiments, it is not intended that each embodiment cover only a single embodiment. Such descriptions of the embodiments are provided for clarity only, and one skilled in the art should consider the embodiments as a whole, and the embodiments in each embodiment can be appropriately combined to form other embodiments as can be understood by one skilled in the art.

Reference documents:

1.Dong XN,Wu Y,Chen YH.2005.The neutralizing epitope ELDKWA on HIV-1 gp41:genetic variability and antigenicity.Immunol Lett.101:81-6.

2.Wang Z,Liu Z,Cheng X,Chen YH.2005.The recombinant immunogen with high-density epitopes of ELDKWA and ELDEWA induced antibodies recognizing both epitopes on HIV-1gp41.Microbiol Immunol.49:703-9.

3.Rerks-Ngarm S,Pitisuttithum P,Nitayaphan S,Kaewkungwal J,Chiu J,Paris R,Premsri N,Namwat C,de Souza M,Adams E.2009.Vaccination with ALVAC and AIDSVAX to prevent HIV infection in Thailand.N Engl J Med.361:2209-2220.

4.Haynes BF,Gilbert PB,McElrath MJ,Zolla-Pazner S,Tomaras GD,Alam SM,Evans DT,Montefiori DC,Karnasuta C,Sutthent R.2012.Immune-correlates analysis of an HIV vaccine efficacy trial.N Engl J Med.366:1275-1286.

5.Nicole L.Yates et al.Vaccine-induced Env V1-V2IgG3correlates with lower HIV-1 infection risk and declines soon after vaccination.Sci Transl Med.2014,6,228ra39.

6.Bruel T,Guivel-Benhassine F,Amraoui S et al.Elimination of HIV-1-infected cells by broadly neutralizing antibodies.Nat Commun 2016；7:10844.

7.Sun,Z.,Zhu,Y.,Wang,Q.,Ye,L.,Dai,Y.,Su,S.,Yu,F.,Ying,T.,Yang,C.,Jiang,S,Lu,L.An immunogen containing four tandem 10E8epitope repeats with exposed key residues induced antibodies to neutralize HIV-1and to activate ADCC reporter gene.Emerg Microb Infect.2016.5:e65.

8.Wang,J.,Tong,P.,Lu,L.,Zhou,L.,Xu,L.,Jiang,S.,Chen,Y.H.(2010).HIV-1 gp41 core with exposed membrane-proximal external region inducing broad HIV-1 neutralizing antibodies.PloS One.6(3):e18233.)

sequence listing

<110> university of Compound Dan

<120> HIV vaccine and method for preparing the same

<130> CP20160446

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 731

<212> PRT

<213> type 1 Human immunodeficiency virus (Human immunodeficiency virus type 1)

<400> 1

Met Arg Val Lys Gly Ile Arg Lys Ser Tyr Gln Tyr Leu Trp Lys Gly

1 5 10 15

Gly Thr Leu Leu Leu Gly Ile Leu Met Ile Cys Ser Ala Val Glu Lys

20 25 30

Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr

35 40 45

Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val

50 55 60

His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro

65 70 75 80

Gln Glu Val Val Leu Glu Asn Val Thr Glu His Phe Asn Met Trp Lys

85 90 95

Asn Asn Met Val Glu Gln Met Gln Glu Asp Ile Ile Ser Leu Trp Asp

100 105 110

Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu

115 120 125

Asn Cys Lys Asp Val Asn Ala Thr Asn Thr Thr Asn Asp Ser Glu Gly

130 135 140

Thr Met Glu Arg Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr

145 150 155 160

Ser Ile Arg Asp Glu Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu

165 170 175

Asp Val Val Pro Ile Asp Asn Asn Asn Thr Ser Tyr Arg Leu Ile Ser

180 185 190

Cys Asp Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Ile Ser Phe Glu

195 200 205

Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys

210 215 220

Cys Asn Asp Lys Thr Phe Asn Gly Lys Gly Pro Cys Lys Asn Val Ser

225 230 235 240

Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu

245 250 255

Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile Arg Ser Asp

260 265 270

Asn Phe Thr Asn Asn Ala Lys Thr Ile Ile Val Gln Leu Lys Glu Ser

275 280 285

Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile

290 295 300

His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly Glu Ile Ile Gly

305 310 315 320

Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala Lys Trp Asn Asp

325 330 335

Thr Leu Lys Gln Ile Val Ile Lys Leu Arg Glu Gln Phe Glu Asn Lys

340 345 350

Thr Ile Val Phe Asn His Ser Ser Gly Gly Asp Pro Glu Ile Val Met

355 360 365

His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln

370 375 380

Leu Phe Asn Ser Thr Trp Asn Asn Asn Thr Glu Gly Ser Asn Asn Thr

385 390 395 400

Glu Gly Asn Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn

405 410 415

Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly

420 425 430

Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp

435 440 445

Gly Gly Ile Asn Glu Asn Gly Thr Glu Ile Phe Arg Pro Gly Gly Gly

450 455 460

Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val

465 470 475 480

Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val

485 490 495

Val Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly

500 505 510

Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Met Thr Leu

515 520 525

Thr Val Gln Ala Arg Leu Leu Leu Ser Gly Ile Val Gln Gln Gln Asn

530 535 540

Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln Arg Met Leu Gln Leu Thr

545 550 555 560

Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg

565 570 575

Tyr Leu Gly Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys

580 585 590

Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys

595 600 605

Ser Leu Asp Arg Ile Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg

610 615 620

Glu Ile Asp Asn Tyr Thr Ser Glu Ile Tyr Thr Leu Ile Glu Glu Ser

625 630 635 640

Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys

645 650 655

Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Lys Trp Leu Trp Tyr

660 665 670

Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Leu

675 680 685

Val Phe Thr Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser

690 695 700

Pro Leu Ser Phe Gln Thr Leu Leu Pro Ala Pro Arg Gly Pro Asp Arg

705 710 715 720

Pro Glu Gly Ile Glu Glu Glu Gly Gly Glu Arg

725 730

<210> 2

<211> 470

<212> PRT

<213> type 1 Human immunodeficiency virus (Human immunodeficiency virus type 1)

<400> 2

Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr

1 5 10 15

Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val

20 25 30

His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro

35 40 45

Gln Glu Val Val Leu Glu Asn Val Thr Glu His Phe Asn Met Trp Lys

50 55 60

Asn Asn Met Val Glu Gln Met Gln Glu Asp Ile Ile Ser Leu Trp Asp

65 70 75 80

Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu

85 90 95

Asn Cys Lys Asp Val Asn Ala Thr Asn Thr Thr Asn Asp Ser Glu Gly

100 105 110

Thr Met Glu Arg Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr

115 120 125

Ser Ile Arg Asp Glu Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu

130 135 140

Asp Val Val Pro Ile Asp Asn Asn Asn Thr Ser Tyr Arg Leu Ile Ser

145 150 155 160

Cys Asp Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Ile Ser Phe Glu

165 170 175

Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys

180 185 190

Cys Asn Asp Lys Thr Phe Asn Gly Lys Gly Pro Cys Lys Asn Val Ser

195 200 205

Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu

210 215 220

Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile Arg Ser Asp

225 230 235 240

Asn Phe Thr Asn Asn Ala Lys Thr Ile Ile Val Gln Leu Lys Glu Ser

245 250 255

Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile

260 265 270

His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly Glu Ile Ile Gly

275 280 285

Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala Lys Trp Asn Asp

290 295 300

Thr Leu Lys Gln Ile Val Ile Lys Leu Arg Glu Gln Phe Glu Asn Lys

305 310 315 320

Thr Ile Val Phe Asn His Ser Ser Gly Gly Asp Pro Glu Ile Val Met

325 330 335

His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln

340 345 350

Leu Phe Asn Ser Thr Trp Asn Asn Asn Thr Glu Gly Ser Asn Asn Thr

355 360 365

Glu Gly Asn Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn

370 375 380

Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly

385 390 395 400

Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp

405 410 415

Gly Gly Ile Asn Glu Asn Gly Thr Glu Ile Phe Arg Pro Gly Gly Gly

420 425 430

Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val

435 440 445

Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val

450 455 460

Val Gln Arg Glu Lys Arg

465 470

<210> 3

<211> 85

<212> PRT

<213> type 1 Human immunodeficiency virus (Human immunodeficiency virus type 1)

<400> 3

Gly Thr Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Lys Asp

1 5 10 15

Val Asn Ala Thr Asn Thr Thr Asn Asp Ser Glu Gly Thr Met Glu Arg

20 25 30

Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp

35 40 45

Glu Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro

50 55 60

Ile Asp Asn Asn Asn Thr Ser Tyr Arg Leu Ile Ser Cys Asp Thr Ser

65 70 75 80

Val Ile Thr Gln Ala

85

Claims

1. An AIDS combined vaccine, which comprises the following components which are respectively inoculated in the following sequence:

(a) DNA expressing HIV envelope protein, wherein the sequence of the HIV envelope protein is SEQ ID NO 1;

(b) the HIV gp120 protein sequence protein, wherein the HIV gp120 sequence is SEQ ID NO 2; and

(c) the polypeptide of the amino acid sequence of HIV gp120V1V2 region, wherein the amino acid sequence of the HIV gp120V1V2 region is SEQ ID NO 3.

2. The AIDS combination vaccine of claim 1, wherein the DNA expressing HIV envelope protein is plasmid expressing HIV envelope protein or virus-like particle of HIV envelope protein.

3. The AIDS combination vaccine of claim 2, wherein the plasmid is pCDNA-JR-FL-Env.

4. The AIDS combination vaccine of claim 1 or 2, wherein the AIDS combination vaccine further comprises pharmaceutically acceptable adjuvants including Freund's adjuvant, aluminum hydroxide adjuvant, MF59, AS02A, QA-21, Corynebacterium parvum, lipopolysaccharide, cytokine, alum, and oil adjuvant or water adjuvant.

5. The AIDS combination vaccine of claim 4, wherein the pharmaceutically acceptable adjuvants used in combination with the components (a), (b) and (c) can be the same or different.

6. A kit for the prevention of aids, comprising:

(1) the AIDS combination vaccine of any one of claims 1 to 5; and

(2) instructions for administering said combined AIDS vaccine.

7. Use of the AIDS combination vaccine of any of claims 1-5 in the preparation of a medicament for the prevention of AIDS.